CMB Exam 1 - Processes

Question 1

extrinsic (receptor-initiated) apoptosis

Answer 1

Death ligand (eg TNF) initiates receptor oligomerization; death domain (DD) recruits adaptors that interact with caspase-8 (forming the death-inducing signaling complex, DISC) leading to autoproteolytic activation of Bcl-2 proteins, caspase-3, etc.

Question 2

intrinsic (mitochondrial) cell death pathway

Answer 2

Mitochondrial integrity is compromised causin release of cytochrome c (and stuff) to initiate apoptosis (regulated by Bcl-2 family). Cytochrome c facilitates interaction of Apaf 1 with the procaspase-9 CARD domain.

Question 3

DNA replication in prokaryotes

Answer 3

First, an initiatior binds to the ori sequence, opening a replication bubble. Then helicase begins to unzip the helix while gyrase (topoisomerase) moves ahead to remove the supercoil and single strand DNA binding proteins hold the strands open. Primase lays the RNA primer for DNA polymerase III to lay the nucleotides. DNA polymerase I removes primers and replaces them with DNA, DNA ligase closes the gaps. To terminate, the polymerase can run into Tus protein, which binds termination sequences.

Question 4

insulin receptor process

Answer 4

When insulin binds tyrosine kinase receptors dimerize and then phosphorylate each other. Next they phosphorylate IRS-1 (scaffold protein). Phosphorylated IRS-1 scaffolding can activate GRB2 and the Ras pathway (alters gene expression), or it can activate PI-3 kinase/ PI-3 kinase phosphorylates PIP2 to PIP3 which activates Akt, triggering the insertion of new GLUT4 channels and activation of glycogen synthase.

Question 5

adenylyl cyclase cascade pathway

Answer 5

Turns ATP into cAMP, which acts on protein kinase A (and other stuff). The activated C portion of PKA can phosphorylate glycogen synthase (to inhibit) and phosphorylase kinase (to activate) which phosphorylates phosphorylase (to activate) which helps turn glycogen to glucose.

Question 6

How does actin bind to adherens junctions?

Answer 6

Contacts between cells are mediated by cadherins and catenins, and stabilized by p120.

Question 7

Describe the contraction process of the contraction ring of mitosis.

Answer 7

Contractile rings form around the middle of two cells in mitosis: MLCK phosphorylates light chains in the cells which does two things: promotes the assembly of myosin into philaments and then increases the myosin catalytic activity to allow contraction to take place.

Question 8

base-excision repair

Answer 8

RECOGNITION: DNA glycosylase excises damaged base (eg deaminated cytosine (U)) leaving an AP site; REMOVAL: AP endonuclease cuts phosphodiester bond and deoxyribose is removed; REPLACEMENT by dna polymerase, REJOINing by dna ligase.

Question 9

nucleotide-excision repair

Answer 9

Involves 7 genes (XPA-XPG): RECOGNITION = XPC which recruits XPA, RPA and TFIIH (contains XPB and XPD); TFIIH unwinds ~25bp bubble. REMOVAL: XPG and XPF/ERCC1 complex are endonucleases and excise ~30 bases. DNA polymerase and DNA ligase replace and rejoin.

Question 10

Transcription-coupled repair

Answer 10

RNA polymerase is stalled by damage and is recognized by CsB at damage. CsB recruits CsA, XPA, RPA and TFIIH, which unwinds. XPG and XPF excise damaged bases. DNA polymerase and DNA ligase replace and rejoin.

Question 11

mismatch repair (prokaryotes)

Answer 11

Possible in the hemimethylated state (strand differentiation). RECOGNITION: MutS recognizes mismatch, recruits MutL which recruits MutH which identifies the daughter strand (thanks to hemimethylation). REMOVAL: MutH is an endonuclease and cleaves up methylated strand at GATC sequence; MutS and MutL work with DNA helicase and exonuclease to excise DNA between mismatch and break. DNA polymerase and DNA ligase fix hole.

Question 12

mismatch repair (eukaryotes)

Answer 12

RECOGNITION: MLH and MSH complex binds to mismatch, identifying the daughter strand via single strand breaks. REMOVAL: The endonuclease cleaves between nick (ss break?) and mismatch, THEN helicase unwinds and exonuclease removes several nucleotides. DNA polymerase and DNA ligase replace and rejoin.

Question 13

Transcription in prokaryotes

Answer 13

RNA polymerase binds the promoter (at -10 and -35 bp upstream) to initiate. Polymerase unwinds 12-14bp DNA, forming open complex. After ~10 nucleotides are added, sigma is released. Stop signal is a polyGC region (hairpin) followed by either polyA or its Rho protein.

Question 14

Transcription in eukaryotes

Answer 14

First, TFIID (includes TATA binding factor TBP and TAF’s) binds promoter, then TFIIB binds TBP and the BRE sequences. RNA pol II and TFIIF bind, and then recruit TFIIE and TFIIH (phosphorylates C terminal domain serine-5 residues), completing the preinitiation complex. The mediator complex is also necessary for initiation.

Question 15

translation initiation in eukaryotes

Answer 15

eIF-2+GTP+fmet-tRNA binds with the 40S subunit. 40S recognizes the 7-methylguanosine cap on the 5’ terminus and then scans downstream until it finds an AUG initiation codon. GTP is hydrolyzed, followed by the dissociation of the eIF’s and the binding of the 60S subunit.

Question 16

translation initiation in prokaryotes

Answer 16

First, amino acid activation via aminoacyl-tRNA synthtases. Then, 30S subunit binds IF-1 and IF-3 to prevent premature binding of tRNA and 50S, respectively. Binding of AUG on mRNA to P site of 30S is guided by the Shine Dalgarno sequence. IF-2+GTP+fmet-tRNA bind to 30S forming the pre-initiation complex, and fmet-tRNA binds to the RNA. The 50S subunit arrives, the GTP is hydrolyzed, and the IF’s all leave. This completes the initiation complex.

Question 17

translational elongation in prokaryotes

Answer 17

EF-Tu complexes with GTP and escorts the AA-tRNA to the A site of the ribosome. GTP is the hydrolyzed (rate limiting step, allowing for proofreading!) and EF-TuGDP and Pi dissociate. EF-TuGTP is regenerated with the help of EF-Ts. mRNA slides 3 nucleotides downstream, and EF-G (aka translocase) uses GTP hydrolysis to move the P tRNA to the E site and the A tRNA to the P site. Binding of new AA-tRNA to A site releases tRNA in E site. Completion is signaled by termination codons: RF1 (UAG, {UAA), or UGA} RF2. RF1 or RF2 hydrolyze peptidyl-tRNA bond, and RF3 initiates the release process.

Question 18

translational elongation in eukaryotes

Answer 18

eEF-1alpha complexes with GTP and escorts the AA-tRNA to the A site of the ribosome. GTP is the hydrolyzed (rate limiting step, allowing for proofreading!) and eEF-1alphaGDP and Pi dissociate. eEF-1alphaGTP is regenerated with the help of eEF-1betagamma. mRNA slides 3 nucleotides downstream, and eEF-2 (aka translocase) uses GTP hydrolysis to move the P tRNA to the E site and the A tRNA to the P site. Binding of new AA-tRNA to A site releases tRNA in E site. Completion is signaled by termination codons UAG, UAA, or UGA. eRF-1 recognizes stop and hydrolyzes peptidyl-tRNA bond and initiates the release process.

Question 19

cotranslational targeting of secretory proteins to the ER

Answer 19

As soon as the “ER” signal sequence emerges from the ribosome it is bound by SRP (signal recognition particle) and escorts the ribosome to the membrane, binding the SRP receptor that is adjacent to the translocon. Once ribosome is tethered, SRP is released. Polypeptide is translated and inserted into membrane: the hydrophobic N-terminus of the protein remains in the membrane, the rest enters the ER. Signal peptidase cleaves sequence.

Question 20

posttranslational translocation into the ER

Answer 20

Chaperones escort completed polypeptides to the translocon where the Sec62/63*BiP complex regulates the entrance of proteins into the lumen. BiP brings them in.

Question 21

Describe the folding process in the ER

Answer 21

Calreticulin is a folding sensor, encapsulates protein/sugars. If it’s correctly folding the glucose is removed and protein is released. If it’s incorrect, glucose gets added back on and goes back and it tries to fold again. If it’s severely misfolded then it gets targeted for the ubiquitin degradation pathway.

Question 22

process of endocytosis

Answer 22

Foreign macromolecules bind to the receptors. Adaptors bind the receptor and to clathrin, which forms the coat and pinches off the vesicle.

Question 23

ubiquitination - degradation

Answer 23

STEPS: Ub is activated following attachment to ubiquitin-activating enzyme (E1). It’s transferred to ubiquitin-conjugating enzyme (E2). Ubiquitin ligase (E3) catalyzes the transfer of ubiquitin to amino group of a lysine residue on the target protein. Polyubiquinated proteins recognized by the 26S proteasome.

Question 24

Explain how mitochondria import proteins.

Answer 24

First, the OM has porins that allow passage of proteins. The protein has an N-terminal signal sequence with multiple + AAs to help it find TOM (and TIM, since the IM is -). Molecular chaperones (Hsp70) losely hold the protein and prevent it from folding so it can cross. Proteins in the TIM complex drve protein import with the help of ATP.

Question 25

PCR: steps

Answer 25

Heat DNA to 95°C to separate strands (heat changes the pH). Cool to 55°C so primers can bind. Warm to 72°C and have Taq polymerase extend the DNA strands. Repeat.

Question 26

M-CDK regulation

Answer 26

One phosphate turns it on, two phosphates turns it off. CAP kinase adds the first phosphate to turn it on, Wee1 kinase adds the second phosphate to turn it off, Cdc25 phosphatase removes the second phosphate to turn it back on.

Question 27

What are the (4) main mechanisms for inducing apoptosis? As in, what happens in the cell to undergo apoptosis?

Answer 27

Activation of caspases, activation of endonuclease, membrane asymmetry, expression of eat-me signal on cell surface.

Question 28

intrinsic (mitochondrial) cell death pathway

Answer 28

Mitochondrial integrity is compromised causin release of cytochrome c (and stuff) to initiate apoptosis (regulated by Bcl-2 family). Cytochrome c facilitates interaction of Apaf 1 with the procaspase-9 CARD domain.

Question 29

extrinsic (receptor-initiated) apoptosis

Answer 29

Death ligand (eg TNF) initiates receptor oligomerization; death domain (DD) recruits adaptors that interact with caspase-8 (forming the death-inducing signaling complex, DISC) leading to autoproteolytic activation of Bcl-2 proteins, caspase-3, etc.

Question 30

What is the order of protein binding in eukaryotic transcription?

Answer 30

TFIID finds spot, then TFIIB, then TFIIF and polymerase, thenn TFIIE and TFIIH. (David Baltimore First Explained HIV reverse transcriptase)

Question 31

Describe the formation of the 3’ end of eukaryotic mRNA

Answer 31

There’s a U- or GU- rich sequence sequence downstream of the gene; this signals endonuclease cleavage at the polyadenylation site. Poly-A polymerase adds the tail.

Question 32

Describe the spliceosome process

Answer 32

The 5’ end of U1 snRNA recognizes the 5’ splice site of pre-mRNA. U2 snRNA recognizes the branch site of pre-mRNA, and binds U1, folding the RNA over. U4/U6 and U5 snRNPs joins the complex and form the intron loop at the branch point.

Question 33

Explain sex differentiation in Drosophila.

Answer 33

mRNA splicing. Females express the SXL gene that helps splice out the UAG gene. Males don’t express the SXL gene, so UAG is included inthe mRNA.

Question 34

adenylyl cyclase cascade pathway

Answer 34

Turns ATP into cAMP, which acts on protein kinase A (and other stuff). The activated C portion of PKA can phosphorylate glycogen synthase (to inhibit), and phosphorylase kinase (to activate) which phosphorylates phosphorylase (to activate) which helps turn glycogen to glucose.

Question 35

phospholipase C pathway

Answer 35

Activated by Gq GPCRs. Cleaves phosphatidyl inosine 2 (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG). DAG activates PKC, which phosphorylates other stuff. IP3 increases intracellular Ca++, which activates calmodulin. Calmodulin activates enzymes like myosin light chain kinase.

Question 36

insulin receptor process

Answer 36

When insulin binds tyrosine kinase receptors dimerize and then phosphorylate each other. Next they phosphorylate IRS-1 (scaffold protein). Phosphorylated IRS-1 scaffolding can activate GRB2 and the Ras pathway (alters gene expression), or it can activate PI-3 kinase/ PI-3 kinase phosphorylates PIP2 to PIP3 which activates Akt, triggering the insertion of new GLUT4 channels and activation of glycogen synthase.

Question 37

Describe cytokine receptor mechanism

Answer 37

Cytokine receptors recruit “non-receptor protein kinases” which dimerize and phosphorylate JAK. JAK phosphorylates STAT, which dimerizes and then can enter the nucleus and act as a transctiption factor.